Drosophila. Mechanistically, the N-terminus of CEP290 directly recruits DAZ interacting zinc finger protein 1 (DZIP1), which then recruits Chibby (CBY) and Rab8 to promote early ciliary membrane formation. Complete deletion of CEP290 blocks ciliogenesis at the initiation stage of TZ assembly, which can be mimicked by DZIP1 deletion mutants. Remarkably, expression of the N-terminus of CEP290 alone restores the TZ localization of DZIP1 and subsequently ameliorates the defects in TZ assembly initiation in cep290 mutants. Our results link CEP290 to DZIP1-CBY/Rab8 module and uncover a previously uncharacterized important function of CEP290 in the coordination of early ciliary membrane formation and TZ assembly.Cilia play critical roles during embryonic development and adult homeostasis. Dysfunction of cilia leads to various human genetic diseases, including many caused by defects in transition zones (TZs), the “gates” of cilia. The evolutionarily conserved TZ component centrosomal protein 290 (CEP290) is the most frequently mutated human ciliopathy gene, but its roles in ciliogenesis are not completely understood. Here, we report that CEP290 plays an essential role in the initiation of TZ assembly in  Dysfunction of cilia leads to various human genetic diseases, including many caused by defects in transition zones (TZs), the “gates” of cilia. A study in Drosophila reveals that the cilia TZ core protein CEP290 coordinates early ciliary membrane formation and TZ assembly; the N-terminus of CEP290 recruits DZIP1, which in turn recruits Rab8 and CBY to promote early ciliary membrane formation. Cilia are microtubule-based organelles that extend from the surface of most eukaryotic cells. These protrusions are important for diverse cellular functions, including cell signaling, sensory perception, cell motility, and extracellular fluid movement –4. In huCilia arise from the distal ends of basal bodies (BBs) that are derived from mother centrioles. Depending on the cell type, cilia form through 1 of 2 pathways: the intracellular pathway and the extracellular pathway ,8. RecenC. elegans have grouped known TZ proteins into 3 functional modules, namely, Meckel–Gruber syndrome (MKS), Nephronophthisis (NPHP), and CEP290 [The TZ is characterized by Y-link–like structures under electron microscopy, which connect proximal axonemal microtubules to the ciliary base membrane and may organize the ciliary necklace composed of annular intramembrane particles on the TZ membrane surface. It has been proposed that the TZ functions as a diffusion barrier to gate the ciliary compartment –18. Dozed CEP290 –22. AlthChlamydomonas to humans [CEP290 cause several ciliopathies ranging from nonsyndromic retinal degeneration, i.e., Leber Congenital Amaurosis (LCA), to syndromic disorders including, Senior–Loken syndrome (SLS), Joubert syndrome (JBTS), MKS, and Bardet–Biedl syndrome (BBS) [CEP290 mutations have been identified in patients, and these mutations cause a wide variety of phenotypes, ranging from isolated blindness to embryonic lethality [CEP290 mutations highlight the multiple and critical roles of CEP290 in cilia. However, the molecular mechanisms of CEP290 in TZ assembly are still not fully understood.CEP290, an important TZ component, is evolutionarily conserved from unicellular o humans ,21,23–25me (BBS) . Over 10ethality . The broDrosophila. Complete deletion of CEP290 blocks ciliogenesis at the stage of TZ assembly initiation in both ciliated sensory neurons and spermatocytes, 2 main ciliated cell types of Drosophila. Further studies reveal that the N-terminus of CEP290 directly interacts with DAZ interacting zinc finger protein 1 (DZIP1) and recruits DZIP1 to the TZ. Interestingly, deletion of DZIP1 results in a phenotype similar to cep290 deletion mutants. Furthermore, we demonstrate that DZIP1 functions upstream of CBY and Rab8 to mediate ciliary membrane assembly. We propose that ciliary membrane assembly is a prerequisite for TZ assembly and that CEP290 coordinates the formation of early ciliary membranes with the assembly of the TZ.Here, we discover that CEP290 is essential for TZ assembly initiation in Drosophila CEP290, we constructed several green fluorescent protein (GFP)-tagged CEP290 truncation constructs and assessed their subcellular localization in various types of cilia in Drosophila: auditory cilia, olfactory cilia, and spermatocyte cilia. It has been proposed that CEP290 bridges axonemal microtubules and ciliary membranes, where its C-terminus contains a microtubule binding domain and N-terminus possesses a highly conserved membrane-binding amphipathic α-helix motif  may be not a null mutant [cep2901 (c.469-1174Del), in which site-specific deletion leads to a frameshift, leaving only 156 aa of the N-terminus , in which a deletion results in a frameshift and C-terminus (aa 1386-end) loss  loss . As expe mutants . Notably reduced . This isat of WT , probablcep2901 and cep290ΔC were severely uncoordinated during walking and flying, and they had severe defects in both touch sensitivity and hearing sensation  CilDrosophila [cep290ΔC mutant, although the signal intensity of MKS1, MKS6, and CBY was significantly reduced  and TZ-related proteins (CBY and DILA/CEP131). CBY and Dilatory (DILA) cooperate to build the TZ in osophila , but theosophila , we obse reduced . However mutants , while tcep290ΔC mutants, the signal of Uncoordinated  was aberrantly elongated in cep2901 mutants  assays  assays . Our EM  mutants . Althoug mutants . Similar mutants . These rTaken together, our results indicate that CEP290 is essential for the initiation of TZ assembly in both sensory neurons and spermatocytes. Of note, without a robust antibody to survey expression to validate levels of CEP290 protein expression within our alleles, we cannot fully unpick how much of our phenotypes are due solely to reduced levels versus requirement for specific domains. We suspect it is likely a combination of both.Drosophila DZIP1 is the ortholog of mammalian DZIP1 and DZIP1L (Drosophila S2 cells and performed coimmunoprecipitation (Co-IP) using GFP-trap beads. Western blots of the GFP-pulldowns confirmed the binding between CEP290 and DZIP1 , in which the C-terminus (aa 357-end) of DZIP1 is lost  regulates the migration of centriole/BB [Drosophila spermatocytes [Drosophila-specific structures called dense fibers that connect the proximal centriole (daughter centriole) to the membrane [Drosophila.From our TEM analysis, we observed that BBs are attached/very close to the plasma membrane in proposed . Howeverriole/BB ,49. But atocytes . In sensmembrane ,52. It iDrosophila model, we demonstrated that Rab8 and CBY-FAM92 module function downstream of DZIP1 and jointly regulate TZ assembly initiation. The fact that rab8; cby double mutants and rab8; fam92 double mutants show synthetic defects suggests that they have synergetic roles in ciliogenesis. It is tempting to speculate that CBY-FAM92 module promotes membrane remodeling and then facilitate the Rab8 and its yet unknown compensatory mechanism regulating the ciliary membrane formation. The data available so far suggest that the function of DZIP1-Rab8 module and DZIP1-CBY-FAM92 module in ciliogenesis is largely conserved from Drosophila to mammals [Drosophila, Dzip1 or Dzip1l knockout (KO) mice have much more severe defects than Cby1 KO mice or Fam92a KO mice, supporting the notion that CBY-FAM92 is not the solely downstream pathway of DZIP1 or DZIP1L [By using the simple  mammals . The int mammals ,37,40,43r DZIP1L ,36,44,54Drosophila model, we demonstrated that TZ core protein CEP290 directly interacts with DZIP1 and controls TZ assembly initiation by regulating DZIP1-mediated early ciliary membrane formation. It has been reported that mammalian DZIP1/DZIP1L interacts with Rab8 and CBY [Dzip1l mutant mice [CEP290-deficient patients [For the first time, using the  and CBY ,37,40, aant mice . Thus, tant mice . Althougant mice , and defpatients . In addiw1118 , elav-GAL4 (BDSC 458), unc2 (BDSC 7424), rab81 (BDSC 26173), Df(3L)ED228 (BDSC 8086), DJ-GFP (BDSC 5417), UASp-YFP-Rab8T22N (BDSC 9780), UASp-YFP-Rab8Q67L (BDSC 9781), and UASp-YFP-Rab8 (BDSC 9782); CG6652-GFP, UNC-GFP, MKS1-GFP and MKS6-GFP were provided by Dr. Bénédicte Durand; nompC-Gal4; UAS-GFP was provided by Dr. Wei Zhang. The flies were raised on standard media at 25°C.The fly strains used in the study are as follows: Drosophila Resources and Technology, Shanghai Institute of Biochemistry and Cell Biology (SIBCB), Chinese Academy of Sciences (CAS). To construct the FBF1-GFP, CBY-GFP, and DZIP1-GFP plasmids, the entire coding sequences (CDSs) with their promoter regions were separately cloned and inserted into the HindIII and BamHI sites of PJFRC2 vectors using a one-step cloning method. To construct the UAS-DZIP1-GFP and UAS-CEP290-GFP plasmids, we cloned the entire CDSs into the BglII and BamHI sites of PJFRC2 vectors. The DILA-GFP, truncated CEP290, and truncated CEP290-GFP plasmids were prepared similarly. We first subcloned the ubiquitin promoter region from pWUM6 into the HindIII and BglII sites of PJFRC2 vector or PJFRC2-APEX vector, and then individually cloned each gene cDNA sequence into the NotI and BamHI sites of the vector. Transgenes of CEP290, DILA, and UNC were inserted at the attP site of the 25C6 locus on chromosome 2, and transgenes of FBF1 and DZIP1 were inserted at the attP site of 75B1 locus on chromosome 3. All CDS fragments were amplified from cDNA reverse transcribed from total RNA. The primers used are listed in Transgenic lines were generated by the Core Facility of http://tools.flycrispr.molbio.wisc.edu/targetFinder). Customized single-guide RNA (gRNA) oligos were synthesized and then cloned into the BbsI site of the pEASY-PU6-BbsI-chiRNA vector. All the constructed plasmids were then injected into vasa-Cas9 embryos by the Core Facility of Drosophila Resources and Technology, SIBCB, CAS following a standard protocol. The mutant fly lines were screened via PCR. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the transcription level. The rp49 gene was used as an internal reference. The primers used are listed in The CRISPR/Cas9 system was used to generate the deletion mutants. Briefly, CRISPR targeting sites were designed using Target Finder . The collected flies were dissected and washed with 1× PBS. The dissected tissues were then placed on slides and squashed with coverslips. The slides were submerged in liquid nitrogen for 1 min, after which coverslips were removed. After incubation in cold methanol (−20°C) for 15 min followed by cold acetone (−20°C) for 10 min, the slides were washed 2 times for 10 min each in 1× PBS at room temperature, and then blocked for at least 1 h in blocking buffer  at room temperature and stained with primary antibodies overnight in a moisture chamber at −4°C. After washing with 1× PBS, the slides were incubated with secondary antibodies for 3 h at room temperature.Testes from juvenile flies were dissected in PBS and placed on slides. The testes were stained in CellMask Deep Red  solution (40 μg/mL) for 5 min at room temperature.Rabbit antibodies against fly DZIP1 (aa 451–737) and CEP290 (aa 292–541) were produced by YOUKE Biotech . Mouse anti-GFP , rabbit anti-GFP , mouse anti-γ-tubulin , and mouse anti-21A6  were also used. The secondary antibodies (1:1000 dilution) were goat anti-mouse Alexa Fluor 488/594 or goat anti-rabbit Alexa Fluor 488/594.n = 50 centrioles or BBs over 5 flies.Most slides were visualized using a fluorescence microscope  or a confocal microscope  with a 100 × (1.4 NA) oil-immersion objective. SIM images were taken using a Delta Vision OMX SR  with a 60 × (1.42 NA) oil-immersion objective. All data analysis was performed using ImageJ . For quantification of signal intensity of TZ proteins, the fluorescence intensity is calculated by subtracting the sum of background signals of the same size from the sum of the signals in the region of interest, 4. The samples were dehydrated using a diluted ethanol series and embedded in epoxy resin. Ultrathin sections (approximately 70 nm) were cut using an Ultracut S ultramicrotome . The samples were stained with uranyl acetate and lead citrate. Electron microscopy observations were made with Hitachi H-7650 transmission electron microscope  at 80 kV.Testis and antenna samples were prepared according to published protocols. Briefly, tissues were fixed in 2.5% glutaraldehyde buffer for at least 24 h and post fixed in OsOw1118 females for 3 days. The fertility ratio was quantified, and the progeny were counted.Single newly enclosed males were crossed with a virgin For the adult climbing assay, 10 flies aged 3 to 5 d were placed in a graduated cylinder. At the beginning of each test, the flies were gently tapped to the bottom of the cylinder. The flies that climbed above the 8-cm height mark in 10 s were then counted, and counting was repeated 10 times. The data for each genotype are the averages of 5 biological replicates.The larval touch assay was performed as previously described . A singlThe larval hearing assay was processed as previously described . In eachDZIP1 was introduced into the pGBKT7 vector , and CEP290-N (aa 1–887) and CEP290-ΔN (aa 888–1978) were individually inserted into the pGADT7 vector (Clontech). The constructs were subsequently transferred into strain AH109  by the LiCl-polyethylene glycol method. Yeasts were grown on SD-Leu-Trp plates at 30°C. After 3 to 4 d of incubation, positive colonies were tested on SD-Ade-Leu-Trp-His plates and incubated for 5 d at 30°C.Drosophila Gateway vector PAWG or PAWH, respectively. GFP was cloned into pAWM as a control. One microgram of total DNA was transfected into S2 cells or HEK293 cells in a 60-mm plate with Effectene . After 48 h, the cells were lysed in ice-cold Co-IP lysis buffer  at 4°C for 45 min and centrifuged at 20,000 × g for 10 min at 4°C. The supernatants were added to anti-GFP nanobody agarose beads , and the beads were tumbled end over end for 1 h at 4°C. After western blotting, the polyvinylidene fluoride (PVDF) membrane was incubated with anti-GFP antibody (Roche) and anti-HA antibody (Invitrogen) or anti-Flag antibody (Sigma). Full scans of the western blots are presented in To construct the DZIP1-GFP, CEP290-N-GFP (aa 1–878), DZIP1-HA, CBY-HA, and Rab8-HA plasmids, we cloned the CDSs into the T22N, and His-Rab8Q67L plasmids, we cloned the CDSs into the pET28a or pGEX-4 T-1 vector. The constructs were transformed into BL21 (DE3) E. coli. The proteins were expressed and purified using His resin or Glutathione Sepharose. The purified His-CEP290-N, His-CEP290-ΔN, His-CBY, His-Rab8, His-Rab8T22N, and His-Rab8Q67L proteins were incubated with either GST, GST-DZIP1-N, or GST-DZIP1-C in binding buffer  overnight at 4°C. After western blotting, the PVDF membrane was incubated with anti-His antibodies (Invitrogen) and stained with Ponceau S. Full scans of the western blots are presented in To generate the GST-DZIP1-N (aa 1–293), GST-DZIP1-C (aa 294–737), His-CEP290-N (aa 1–887), His-CEP290-ΔN (aa 888–1978), His-CBY, His-Rab8, His-Rab8t tests. .The results are presented in all figures as the means and SDs. Statistical analyses were performed with two-tailed unpaired Student S1 Figcep2901 mutants by PCR of CEP290 fragment spanning the deletion region using whole fly genomic DNA. The amplification products were 1,754 bp long for w1118 and 929 bp long for cep2901 mutants. (B) RT-PCR analysis of the splice isoforms and transcription level of cep290 gene in cep290ΔC mutants. Amplification of the C-terminal half of cep290 CDS (2662–5937) did not show other splice forms caused by the deletion mutation. Semiquantitative RT-PCR analysis of the transcription level of cep290 CDS using primer F2 and R1 showed that the transcription level was reduced in cep290ΔC mutant, probably due to nonsense-mediated mRNA decay. Housekeeping gene rp49 was used as the internal control. (C) cDNA sequencing verification of cep290ΔC mutant. (D) Upper panel: Immunofluorescence staining with an anti-CEP290 N (aa292-541) antibody confirmed that the CEP290 N-terminal signal was reduced in cep290ΔC mutants but completely lost in cep2901 mutants. The corresponding relative fluorescence intensity was quantitatively displayed in lower panel. The bars and error bars represent the means and SDs, respectively. n = 50 centrioles over 5 flies. Scale bars, 1 μm. (E) Cilia-related behavior analysis of cep290 mutants. Both cep290ΔC and cep2901 mutant flies have severe defects in hearing (left panel) and touch sensitivity (right panel). Expression of CEP290 rescued these defects. For the hearing assay, 5 larvae as a group, and at least 5 groups of flies were tested. For the touch sensitivity assay, n = 50. (F) Testes of WT flies and cep290 mutants. The mitochondrial protein DJ was used to label sperm cysts. Compared to those in WT flies and cep290ΔC mutants, sperm cysts in cep2901 mutants were severely defective in elongation. The arrow indicates that the cysts failed to elongate. Bar, 200 μm. (G) Male fertility assay in WT flies and cep290 mutants. We first rescued the severely uncoordinated phenotype of cep290 mutants by expressing CEP290 driven by elav-GAL4. The percentage of fertile males was then quantified, and the number of offspring was calculated. cep2901 males were completely infertile, while more than 65% of cep290ΔC males were fertile. n = 50. (H) EM images of testis cross sections from WT flies and cep2901 mutants. There were 64 spermatids in each cyst in WT flies, whereas axonemes were almost completely lost in cep2901 mutants. The arrows show defective axonemes. (I) EM images of longitudinal sections of auditory cilia in WT flies and cep290 mutants. Bars, 200 nm. Numerical data for panels D, E, and G can be found in the file rp49, ribosomal protein 49; RT-PCR, reverse transcription-polymerase chain reaction; TZ, transition zone; WT, wild-type.(A) Genotyping of (TIF)Click here for additional data file.S2 FigDrosophila DZIP1 is orthologous to both DZIP1 and DZIP1L. (C) Schematic representation of the protein structure of Drosophila DZIP1 and human DZIP1 and DZIP1L. All share highly conserved domain that includes Dzip_like-N and ZnF_C2H2. (D) Table showing the sequence similarity and identity between Drosophila DZIP1 and human DZIP1 and DZIP1L.(A) DZIP1 is evolutionarily conserved in Holozoans. There is only 1 DZIP1 gene in invertebrates, while there are 2 genes in vertebrates, DZIP1 and DZIP1L, possibly because of gene duplication. (B) Phylogenetic tree of DZIP1. (TIF)Click here for additional data file.S3 Fig(A) DZIP1 did not interact with CEP290-ΔN (Δ aa 1–887) in the GST pull-down assay. (B) The GST pull-down assay was used to narrow down the interaction region between CEP290 and DZIP1. DZIP1-N (aa 1–293) interacts with both CEP290 N (aa 401–650) and CEP290 N (aa 651–887), and DZIP1-C (aa 294–737) interacts with CEP290 N (aa 651–887). Uncropped immunoblots can be found in (TIF)Click here for additional data file.S4 FigDrosophila. Upper panel: DZIP1-GFP; lower panel: anti-DZIP1. DZIP1-GFP and anti-DZIP1 staining showed similar localization patterns. No DZIP1 was present on centrioles in spermatogonia. DZIP1 began to appear on the tips of centrioles in early spermatocytes. In round spermatids, DZIP1 migrated together with the ring centriole to the tip of flagella. The centrioles/BBs were labeled with γ-Tubulin (red). Bar, 2 μm. (B) DZIP1 localized to the ciliary base in both auditory and olfactory cilia. Ciliary bases were labeled with 21A6 (red). Anti-DZIP1 staining showed a localization pattern similar to that of DZIP1-GFP. Bar, 1 μm. (C) 3D-SIM images of DZIP1 localization in spermatids, DZIP1 colocalized with CBY at the ring centriole, but did not extend like MKS1. Bar, 500 nm. (D) 3D-SIM images of spatial relationship between TF marker FBF1 and TZ core protein MKS1 in various types of cilia. Notably, there is a gap (arrows) between FBF1 and MKS in sensory cilia but not in spermatocyte cilia. Bar, 500 nm. 3D-SIM, three-dimensional structured illumination microscopy; BB, basal body; CBY, Chibby; DZIP1, DAZ interacting zinc finger protein 1; FBF1, Fas binding factor 1; GFP, green fluorescent protein; MKS, Meckel–Gruber syndrome; MKS1, Meckel syndrome type 1; TF, transition fiber; TZ, transition zone.(A) Subcellular localization of DZIP1 during spermatogenesis in (TIF)Click here for additional data file.S5 Figdzip1 deletion mutant. Schematic of the dzip1 gene. The gRNA target sites are represented by scissors. dzip1ΔC has a deletion of cDNA from nt 1,069 to 1,529, resulting in a frameshift and a premature stop codon and leading to the loss of the C-terminus. (B) Genotyping of the dzip1 mutant. The sizes of the PCR products are as follows: 1,070 bp in w1118 flies and 609 bp in dzip1ΔC flies. (C) Anti-DZIP1 staining confirmed that DZIP1, at least the C-terminus of DZIP1, was completely lost in dzip1ΔC mutants. Bars, 1 μm. (D) dzip1ΔC mutant flies showed defects in hearing and touch sensitivity, but expression of DZIP1-GFP driven by its own promoter rescued these defects. The retraction index is shown as the median and interquartile range. Numerical data can be found in the file n = 50. (E) nompC-Gal4; UAS-GFP was used to mark auditory cilia. Cilia completely disappeared in dzip1ΔC mutants. (F) EM images of cross sections of cilia showing that no axonemes existed in dzip1ΔC mutants. The black arrows indicate ciliary axonemes in WT flies. Bars, 200 nm. DZIP1, DAZ interacting zinc finger protein 1; EM, electron microscope; GFP, green fluorescent protein; gRNA, guide RNA.(A) Generation of the (TIF)Click here for additional data file.S6 FigQ67L-GFP colocalized with DZIP1 at the ciliary base in auditory cilia and olfactory cilia. Notably, Rab8Q67L-GFP was frequently observed in cilia. Bar, 1 μm. (B) 3D-SIM images of the colocalization of Rab8Q67L-GFP and DZIP1 at the base of auditory cilia. Rab8 Q67L-GFP surrounded DZIP1 at TZ and expanded into cilia. Bar, 1 μm. (C) DZIP1 was critical for Rab8-GFP localization at the ciliary base of olfactory cilia. Live imaging showed that Rab8-GFP exhibited a clear 2-dot pattern at the ciliary base in WT flies, but the signal was dispersed in dzip1 mutants. Interestingly, these dispersed signals completely disappeared in our immunofluorescence assay, most likely due to fixation. Bars, 5 μm. (D) DZIP1 was required for Rab8 Q67L-GFP localization at the ciliary base of auditory cilia. Bar, 1 μm. (E) Rab8-GFP was completely lost from the ciliary base of both auditory cilia and olfactory cilia in cep2901 mutants, indicating that CEP290 is essential for Rab8 localization. Bar, 1 μm. (F) Cilium morphology was normal in rab81 mutants. (G) rab8 mutants exhibited normal hearing responses and normal touch sensitivity. Numerical data can be found in the file (A) Rab8-GFP and Rab8(TIF)Click here for additional data file.S7 FigThe interaction between human CEP290 and DZIP1L was analyzed using an immunoprecipitation assay. Human CEP290-N-GFP and Flag-DZIP1L were transiently transfected into HEK293 cells; 48 h later, cells were lysed and subjected to Co-IP using GFP-trap beads. Uncropped immunoblots can be found in (TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Raw Image(TIF)Click here for additional data file.S2 Raw Image(TIF)Click here for additional data file. 15 Jan 2020Dear Dr Wei, Thank you for submitting your manuscript entitled "CEP290 is essential for the initiation of transition zone assembly" for consideration as a Research Article by PLOS Biology.Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission out for external peer review. Nevertheless, we will need to be convinced by the reviewers that the novelty and significance of the findings is sufficient for publication in the journal. A better characterization of the role of vesicle and membrane assembly, which is a bit confusing in the study at the moment, and which other factor(s) in adition to Rab8 might be implicated in this process might be required. Please note that before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.Please re-submit your manuscript within two working days, i.e. by Jan 17 2020 11:59PM.https://www.editorialmanager.com/pbiologyLogin to Editorial Manager here: http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF when you re-submit. During resubmission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review. plosbiology@plos.org if you have any queries relating to your submission.Feel free to email us at Kind regards,Ines--Ines Alvarez-Garcia, PhDSenior EditorPLOS BiologyCarlyle House, Carlyle RoadCambridge, CB4 3DN+44 1223–442810 3 Mar 2020Dear Dr Wei,Thank you very much for submitting your manuscript "CEP290 is essential for the initiation of transition zone assembly" for consideration as a Research Article at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by four independent reviewers.The reviews of your manuscript are appended below. As you will see, the reviewers find the work very nicely done and potentially interesting, but they also raise concerns regarding the novelty of certain aspects of the findings They each suggests experiments to confirm and extend some of the findings, like the role of Rab8 in TZ assembly, the interaction between CEP290 and DZIP1/Rab8 or exploring thoroughtly the phenotype of the CEP290 mutants. After discussing the reviews with the academic editor, we think that in general elucidating the Rab8 axis will be necessary for us to consider the manuscript further for publication.I regret that we cannot accept the current version of the manuscript for publication. We remain interested in your study and we would be willing to consider resubmission of a comprehensively revised version that thoroughly addresses all the reviewers' comments. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript would be sent for further evaluation by the reviewers.We appreciate that these requests represent a great deal of extra work, and we are willing to relax our standard revision time to allow you six months to revise your manuscript.We expect to receive your revised manuscript within 6 months.plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.Please email us  and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/*PLOS Data Policy*http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information . For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5Please note that as a condition of publication PLOS' data policy  such that it can be cited independently in the future. For instructions see: Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Ines--Ines Alvarez-Garcia, PhDSenior EditorPLOS BiologyCarlyle House, Carlyle RoadCambridge, CB4 3DN+44 1223–446970---------------------------------------------------------------Reviewers’ commentsRev. 1:In this manuscript, Wu et al. investigate the role of Cep290 in ciliogenesis and transition zone (TZ) assembly in Drosophila. By analyzing a novel null mutant of Cep290, they find that the Cep290 N-terminal domain is essential for TZ formation and for persistent membrane association of centrioles in spermatids. These defects are proposed to be due to a newly described interaction between Cep290-N and Dzip1 that is necessary for TZ localization of Dzip1. Lastly, the authors show that Dzip1 cooperates with Cby and Rab8 to promote ciliary membrane formation.Overall, the data are of high quality and clearly presented. The significance of the findings is partly diminished by a number of studies in Drosophila and other organisms that have explored the roles of Cep290, Dzip1, and Cby in transition zone formation. However, the present work fills in important details (such as the novel connection between Cep290 and Dzip1) and carefully examines ciliary phenotypes in several Drosophila cell types. These results are thus likely to be of strong interest to the cilia field . The conceptual advance is somewhat more limited, and thus the significance to a broader audience is less clear. However, based on the overall high quality of the data and the new molecular insights into transition zone formation, I believe a revised manuscript could be a good candidate for publication in PLoS Biology.Major points:1. While a variety of experimental approaches are employed to demonstrate binding between Cep290-N and Dzip1, a relatively large fragment of Cep290 is used (aa 1-650). This region has previously been shown to have various important functional properties , and thus it is possible that the various phenotypic defects attributed to loss of Cep290-N are not due to loss of Dzip1 binding per se but due to disruption of one or more of these other functional elements (in fact the phenotypes seen in Cep290-l mutants that are not present in Cep290-deltaC could be due to any elements within aa 1-1385). This concern is partly mitigated by the similarity of phenotypes seen in Cep290-l and Dzip1 mutants, but it would be helpful if the authors can narrow Cep290's Dzip1 binding region more precisely and analyze ciliary phenotypes when Dzip1 binding is more selectively disrupted (e.g. by rescue of Cep290-l with such a targeted Cep290 mutant). At a minimum this issue should be discussed in more detail in the manuscript.2. A key point in the manuscript is that the N-terminus of Cep290 has key roles in transition zone formation. This conclusion relies on the more severe phenotypes observed in the Cep290-l mutant than the Cep290-deltaC mutant. However, the predicted frameshift produced by the Cep290-deltaC allele is not experimentally verified, and the possibility of alternative splice forms and/or altered transcript stability is not considered. Given the key conclusions based on this mutant, it would be very helpful if the authors could better characterize precisely what form(s) of Cep290 are expressed in this mutant and at what levels relative to wildtype.Minor points:1. Is there any evidence from the authors or others indicating that the reported connection between Cep290 and Dzip1 might be conserved in other species? Similarly, can the pathogenic features of any disease-associated CEP290 mutants potentially be attributed to disruption of the Cep290-Dzip1 interaction? Experiments examining these points may be beyond the immediate scope of this paper but could make for an impactful addition; at minimum further discussion of these points would be helpful.2. There are some confusingly worded sentences and grammatical errors throughout the text (particularly in the last section of the Results).Rev. 2:The transition zone is a particular structure of the ciliary base which plays many roles in cilia assembly and function. Defects in TZ assembly lead to life threatening ciliopathies. Cep290 is a core component of the TZ and has been shown to act upstream of the TZ assembly program in many organisms, but it's precise function in Drosophila is only partially investigated and how it recruits other TZ proteins is not clear.In this manuscript, the authors describe the first Drosophila cep290 complete null mutant and show that Cep290 plays, like in many other organisms, a critical function in the initiation of ciliogenesis and TZ assembly. They demonstrate for the first time that Cep290 can be recruited to the basal body by its N- or C-terminus domains and that Cep290 directly interacts with Drosophila Dzip1. They show that Dzip1 interacts with Cby a key component of TZ assembly in Drosophila and also with Rab8. Their work also investigates if Cby and Rab8 cooperate to build the drosophila TZ.Whereas the manuscript conclusions on Cep290 are well supported by the observations, the role of Rab8 in TZ assembly is not clearly demonstrated and the authors' conclusion is largely overstated. In particular, the phenotype of the rab8 mutant is weakly documented and does not support their conclusion. To be able to conclude on Rab8 function in TZ assembly in Drosophila, the authors should at least address the following points:-show the rescue of the phenotype of cby; rab8 double mutant by Rab8-GFP and, even more demonstrating, not by Rab8T22N.-the sole phenotype described for the cby; rab8 double mutant is a reduction in MKS1-GFP at the TZ? Is this the only phenotype observed for this double mutant? Is there any consequence on behavioral assays? What is the testis phenotype of the rab8 mutant alone and the double cby; rab8 mutant? Is it consistent with a function in TZ assembly? My opinion is that the results on rab8 are much too preliminary to support a role of Rab8 in TZ assembly and actually reduce the impact of the other conclusions that are very interesting.The other following points also need to be addressed before publication:Major points :-The authors nicely show that basal bodies (BB) in cep290 mutants do not build any TZ and are, at best, just apposed to the plasma membrane. This result was already proposed by Jana et al., 2018 using RNAi. However, it is not clear why the authors' conclude that the basal bodies are docked: in particular do the authors observe a thickening of the plasma membrane and connections between the membrane and the BB, as described by Gottardo et al, 2018? How many centrioles were observed by EM? Are they all apposed to the plasma membrane ? On Figure 2G : one of the two basal body does not seem to be docked to the plasma membrane: can this be explained by the level of the section? Do the authors have other sections of this same centriole?-The authors analyze the distribution of different Cep290 truncated fragments fused to GFP. This analysis is apparently done in WT conditions where the endogenous protein is present, what happens in cep2901 rescue conditions ? Could the difference in the distribution of Cep290 truncated fragments just reveal differences in competition behavior between the truncated and Wt protein?Minor points :Overall the IF images of the sensory cilia are of poor resolution. In particular the images of sensory neurons in 1B, 1C, 4F, 5B and D.Figure 1. The authors only show one example of the distribution of the different Cep290-GFP fragments: is the distribution homogenous between centrioles? Please quantify the length on several centrioles to show the distribution.Figure 2C : Quantify the fluorescence intensity in cep290deltaC mutant compared to WT: is there any effect of deleting only the C-terminus, as the flies are hypofertile, and taking into account that mecH alleles show defects in TZ integrity in the testes .Figure 4D: it was previously shown in Jana et al, 2018, that Cep290 radially extends from the center to the periphery of the TZ, depending on whether GFP is fused to the N-ter or C-ter of the protein. This should be taken into account in the interpretation of the high resolution imaging observations.Page 6: the authors propose that expressing the N-ter-Cep290 fragment has a dominant negative effect : are all transgenes inserted on the same platform as this is not explained in the methods section? Thus, in all the experiments based on the expression of these truncated forms, could there be an effect of the expression levels?Page 7: The authors propose that the C-ter and N-ter domains have an inhibitory effect on their localization and cite Drivas et al. to support their hypothesis. However this latter article shows that overexpressing the N or C-ter domain of human CEP290 leads to increased ciliogenesis in cells. This is completely different to what the authors observe here in their manuscript and is misleading.Figure 7 and discussion: there is an apparent confusion in the text and in the figure between what the authors call dense fibers on page 20, which in the referred publications correspond to lateral connections of the proximal (daughter) centriole and the dendrite membrane and what is called "thin fibers" described in Gottardo et al. 2018. These are completely different structures that likely have completely different roles and Figure 7 must be corrected to "thin fibers" as described in Gottardo et al. The EM images of this manuscript do not show that these connections are still present in cep290 mutants. In addition, to my knowledge, ciliary vesicles have not been described on Drosophila EM images, thus parts of Figure 7 are speculative.At many occurrences, the conclusions appear to be overstated:Page 8, end of second paragraph :« Probably its N-terminus alone » this is likely overstated. It shows that the N-terminus is required but not sufficient as the C-terminus truncated Cep290mecH mutants do have a TZ gating defects .Page 13: "DZIP1 did not affect the initial docking of the BB": same comment as above for the cep290 mutant. What are the arguments to say that the BB are docked and not only apposed to the plasma membrane?Page 20: second paragraph: "suggests that they have synergistic roles in early ciliary membrane formation". The manuscript only shows that MKS1 is affected not that there are defects in early ciliary membrane formation.Last paragraph of the result section: the last sentence is highly speculative and should be removed.Some of the results obtained for dzip1 mutant confirm recently published results by Lapart et al, 2019. This should be stated in the result or discussion section.As well, some observations of the cep290 mutants confirm previous published results by Basiri et al., Jana et al. and Lapart et al., and this should be recalled accordingly in the text.Page 21 "regulating DZIP1 mediated early ciliary membrane": incomplete sentence.Rev. 3:In this manuscript, Wu et al. report that the N-terminal region of CEP290 plays an essential role in the initiation of transition zone assembly of the cilia in in neurons and spermatocyte of Drosophila. They demonstrate that the N-terminus of CEP290 directly interacts with DZIP1 and recruits DZIP1 to transition zone, where it recruits CBY and RAB8 to assemble the ciliary membrane. The authors propose that CEP290 coordinates the early ciliary membrane formation and transition zone assembly.Overall, this work is of importance to the ciliogenesis field because little is known about the regulation of early ciliary assembly events. Requirements for CEP290 in transition zone assembly and function has been investigated in several model systems including Drosophila . Importantly, CEP290 has previosuly been reported to act upstream of DZIP1 recruitment - which was not mentioned in this paper. Moreover, associations with Rab8 function have been reported and were for the most part noted in this manuscript with exception a paper in 2008 by Kim et al. Thus, the main findings from this work largely relate to the identification of interactions for CEP290-DZIP1 and DZIP1-CBY/Rab8. However, the interaction between DZIP1 and Rab8 and CBY seem rather weak compared to the input. Another concern I have is that conclusions drawn about TZ structure and protein localization may be over interpreted from the provided SIM imaging. The quality/resolution of these images seems lower than what has been published by others for the TZ in Drosophila.Additional concerns/questions/suggestions are listed below.1. The C-terminus of CEP290 has microtubule binding domain while the N-terminus has membrane binding motif. Here the authors show that the N-terminus alone localizes to the transition zone . How might this fragment be anchored at the transition zone and basal body? Understanding this question could help enhance the mechanistic understanding of this work.2. The 21A6 antibody recognizes two domains longitudinally in auditory neurons. Which domain is shown in Fig. 1B, 4F and 5B? What are the directions of the cilia in these figures? Showing un-cropped images may help to predict the position of the cilia in these cells.3. What are the stages of the spermatocyte in Fig 2C? What structure is the elongated UNC signal related to when the ciliary bud formation is blocked in CEP290 null cells as shown in Fig. 2C and 2F?4. The localization of CG6652 in cep290 null cells is very different comparing to wt and delta C cells in Fig. 2D. The authors claim that this is abnormally extended axoneme. But the distribution of CG6653 does not look like an axoneme. Electron microscopy analysis would be necessary to confirm this structure.5. The association between basal bodies and plasma membrane was not defective in later spermatocytes and spermatids in CEP290 dC mutant cells . The transition zone can be assembled too . Are the cilia buds formed in these cells comparing to wt and null cells as in Fig. 2F and 2G?6. The authors indicate that DZIP1 occupied the gap region between FBF1 and MKS1 in Fig. 4E and S3E. However, there is still a gap between DZIP1 and FBF1 in Fig. 4D auditory neurons. Are these representative images?7. It would be helpful to note in the figure legends that some of the blots are antibody staining while other are are Poncuau S. stained for clarity.8. Showing images of the whole auditory and olfactory systems can help confirm signal from background. The cropped area in images in Fig 1BCD, Fig.3C, Fig. 4F and Fig. 5AB are too small.Rev. 4:The manuscript Wu et al. is a very carefully crafted, well executed and powerful genetic analysis of the role of well-known transition zone factor CEP290 in assembly of different types of cilia in Drosophila. Coupling powerful fly genetics, with rapid transgenics and reporters and quantitative imaging, the authors have created an elegant study into how CEP290, a key human ciliopathy gene, is mechanistically driving assembly of the transition zone in these highly modified cilia types. Indeed, understanding distinct and overlapping properties of the transition zone of different cilia types is key towards understanding the clinical pleiotropy observed in human ciliopathy patients in terms of symptom severity and penetrance between different tissue types. As such the study by Wu et al. is timely and of general interest to an audience like that of PLoS Biology. The tangible and highly subjective aspect of novelty of this study has been recently dinged by the Lampard et al in eLife (December 2019) from the Durand lab, where they looked at the same problem in the same system from a slightly different perspective, focusing on DZIP1 and FAM92, as well as CBY and of least focus CEP290. I believe the studies are complementary, the findings by Wu et al supporting and extending on those in the Lampard study- there is something important to be said for reproduction/confirmation of findings. However, given this and in light of the ethos of PLoS Biology to publish 'outstanding scientific significance', I feel this is somewhat an editorial issue for PLoS to decide where this one falls. Indeed, journals like eLIFE have a clear policy on publishing 'scooped' results of high quality and scientific significance, even if the novelty has been compromised. My gut feeling is it is better placed there.Scientifically I can comment- as shown below. Generally, it is a well-written paper, although many of the figures are too busy/too many panels such that details cannot be seen but this is not fatal. It is an excellent example of genetics, imaging and biochemistry to carefully phenotypically and structurally dissect cellular process- I think it is very elegant in its execution and very thorough, very strong in its analysis. There is a propensity in the discussion to overextend speculation in the mammalian system for some of the points, but I will address them below.Major points:https://imagej.nih.gov/ij/docs/guide/146-9.html- about 10% of your male audience may be colorblind). Also the dark blue/indigo of the CellMask in 2E, 4H-I and 5F is difficult to discern from the black background- consider magenta or something bright.1. Accessibility: Many of the figures are too complex- it is very difficult to see details/labels even at their current full page size . Also as most of the panels have only two dyes/fluorophores- I would strongly urge the authors to consider a color blind palette  which causes a premature termination codon resulting from a frameshift, in the 5th last exon of a very large gene- not being a null allele and instead being a milder loss-of-function allele due to remaining action of the C-terminally truncated protein . Showing my mammalian bias, one would think this would subject the transcript to nonsense mediated decay, instead of protein truncation. Although speculated this truncated protein species exists, it is never investigated. I know that antibodies are difficult for Drosophila studies but some RNA/semi-quantitative PCR to look at levels and altered splice variants could lead credence to this- and that the cep290l and cep290�C lines generated here by genome editing, where larger on-target 'off-target' effects like inversions or larger deletions, make a phenotype seem more severe. Similarly, the transgenic over-expression some of the GFP CEP290 variants result in defective motility where the N-terminal CEP290 'may play a dominant negative role'- if this was true then heterozygous carriers for the cep290�C allele would have a similar uncoordinated, motor phenotype which they do not. Indeed according to the Basiri paper 'cep290mecH homozygotes (cep290) and hemizygotes (cep290/−) cannot stand….cep290mecH heterozygotes (cep290/+) and rescue (Cep290 and Cep290-GFP) are normal.' The lines of argument here are contorted. A safer approach would be like the Lampard paper uses an enhancer trap line- and just says it is a strong hypomorph and has a similar phenotype- loss of FAM92 and DZIP from the transition complexes, and '76% of aberrant axonemal growth, suggesting that basal body to membrane attachment is compromised in this mutant'. Genetics is complicated and sometime a rabbit hole not worth completely going down to prove your alleles are in fact nulls. Indeed in the clinical setting, we know that generally the severity of CEP290-associated phenotypes seems to correlate with the residual amount of functional protein that a mutant allele could produce - something you have not looked at here rather tie it to domains. This is a weakness for me.Minor points:1. Awkward and inexact: Abstract 'Cilia play critical roles in organ generation and maintenance' consider 'during embryonic development and adult homeostasis'.2. Introduction, p3, first line- typo '…that extend from the surface of most eukaryotic…'.3. Introduction, p4, second line- typo '…which connect proximal axonemal…'.4. Introduction, p4, second line Long sentence consider splitting- 'to the ciliary case membrane. These have been proposed to organize… on the membrane surface. The transition zone functions…'.5. Introduction, p4. Second paragraph, third line. Italicize gene 'CEP290' and consider rewording from list of disorders to a more informative statement for non-expert readers like '…several ciliopathies ranging from non-syndromic retinal degeneration  to syndromic disorders including Senior-Loken syndrome (SLS),…'6. Introduction, p4. Second paragraph, missing text. '…diseases and phenotypes associated with CEP290 mutations highlight the multiple and critical…'. Gene names should always be italics.7. Introduction, p.5- inconsistency. I may be missing something but the statement 'We proposed that ciliary membrane assembly is a prerequisite for TZ assembly initiation' seems to be not what it is shown in your summary figure 7- where binding of CEP290 to apical membrane and recruitment of TZ facilitators DZIP and CBY occur before recruitment of Rab8/ciliary membrane vesicles. To the model is the inverse of this 'We proposed that TZ assembly initiation is a prerequisite for ciliary membrane assembly…'8. Results, p.5, last paragraph, missing text. '…microtubules and ciliary membranes, where its C-terminus…'. Consider referring to Figure 1A here.9. Results, p.5/6- clarity. Consider emphasizing constructs are overexpressed in wild type flies. Consider removing dominant negative statement at end of paragraph (see above).10. Figure 1 A-D- do you know that all these GFP constructs are expressed- the lack of staining at the basal body of cilia for CEP290-M could simply be due to unstable protein. Also- I am perplexed: Figure 1B vs 1E- why such a different look to the cilia localizations between modalities- the confocal has two closely aligned structures and the SIM data only has one a field of view that is only double the magnification of the first, according to scale bars. Maybe a statement clarifying this is needed in the legends.11. Results, p.7, for transparence- add reference to previously published alleles for cep290. 'Similar to previously reported alleles, both cep2901 and cep290�C were uncoordinated….. .12. Figure 2A- for clarity, consider annotating where cep290mecH falls. Also explicitly add into the figure legends that these are predicted protein sizes- both of your CRIPSR alleles (and cep290mecH) may be subject to NMD and thus have little protein of any size to speak of.13. Results, p.9, last paragraph- clarify. '…leading to loss of cilia/TZ' as the paragraph is written these structures never form- consider 'completely blocked, such that cilia with transition zones never form'. The following sentence may need some gentle reworking for non-fly enthusiasts, '…resulting in subsequent abnormal elongation of membraneless ciliary axonemes within the cytoplasm'.14. Results, p.10, typo, first sentence. '…the initiation of TZ assembly, spermatogenesis….'.15. Results, p.11, typo, second sentence. '…intensities were severely reduced compared to….'.16. Results, p.11, emphasis. Tone down final statement first paragraph- ' …in sensory neurons, the CEP290 N-terminus may be essential for initial TZ assembly, whereas TZ elongation or maturation may involve a tissue-specific requirement for the C-terminus of CEP290.'17. Results, p.11, typo. '… we employed a yeast two hybrid….' Also- 'Drosophila DZIP1 is the ortholog of mammalian DZIP1..'18. Results, p.11, clarification. What is the CEP290-�N construct? What is in it is never detailed anywhere and is confusing with what is outlined in figure 1A.19. Results, p.12, clarification. 'While the role of Drosophila DZIP1 in ciliogenesis was unknown when we started this project, a very recent publication has confirmed our findings .'20. Results, p.13, typo. '…completely lost but CEP290 persisted…'. Also 'Furthermore, similar to cep2901 mutants…'21. Results, p.14, typo. '… C-terminus may have tissue-specific roles in regulating…'.22. Results, p.17, typo. '…compensate for the loss of Rab8, as has been reported for Rab8 and Rab10 in mammalian ciliogenesis…'.23. Results, p.17, clarify. I was confused by the statement '…then facilitate the Rab8- and its redundant player- mediated…'. Consider '…then facilitate the Rab8- and its yet-unknown compensatory mechanism regulating…'.24. Discussion, p.18, see point 7. '…TZ assembly is completely blocked in dzip1 mutants suggests that early ciliary membrane formation is a prerequisite for TZ assembly initiation'. Again I think this is the opposite of what your data shows- TZ assembly predates early ciliary membrane formation in your analysis.25. Discussion, p.18, careful. The discussion about CEP290 mutations in human patients- there are retinal specific isoforms for many key RP genes which are believed to be the reason for the syndromic versus non-syndromic divide for some of these clinical phenotypes. This should be referenced. Again although the genotype-phenotype correlation is challenging, in general it is believed that the severity of CEP290-associated clinical phenotypes appears to reflect the residual amount of functional protein produced by a mutant allele . This current study does not address any of this- but puts it down to protein domains- which should remain an interpretation of the results, given it could be down to just protein levels.26. Discussion, p.19, typo. 'In Drosophila, the centriole…'. Also take out the '(our unpublished data)' , either you show it or don't mention it at all.27. Discussion, p.20, typo. '…understand the mechanisms of…'.28. Discussion, p.20, clarify (see point 23). Change '…then facilitate the Rab8- and its redundant player- mediated…'. Consider '…then facilitate the Rab8- and its yet-unknown compensatory mechanism regulating…'.29. Discussion, p.20, nomenclature. Mouse alleles should be title case italics- Dzip1 or Dzip1l. See final paragraph too. There is also a typo here- mice is plural so should be 'have much more severe…'. Same for Cby and Fam92a. Within species, they are not orthologs, they are paralogs. Final sentence in paragraph change to '…the existence of so many paralogs complicates the study of their genetic relationship'.30. Discussion, p.21, missing word. '….regulating DZIP1-mediated early ciliary membrane WHAT?….'.31. Methods, p.24, add information. The microscopy and image analysis- how was the quantitative imaging done- how many flies, how many basal bodies/TZ to get traces'.32. Acknowledgements, p.35, typo. Change to '…for their technical support'.33. Figure Legends, 4 title, clarify. '…with the N-terminus of CEP290, and dzip1�C deletion mutants…'34. Supplementary Figure legends, p.42. Clarify PCR is of what- genomic DNA? In figure S1E- there is a typo throughout with your driver- should be elav-GAL4 not ealv. Figure S1- add in what acronym DBB and PBB mean.35. Supplementary Figure legend 2, p.43. There is a typo in the figure for H. Sapiens several times.36. Supplementary Figure legend 3, p.44. Typo '…to the tip of the flagella.' 15 Sep 2020AttachmentResponse to Reviewers final(1).docxSubmitted filename: Click here for additional data file. 18 Nov 2020Dear Dr Wei,Thank you for submitting your revised Research Article entitled "CEP290 is essential for the initiation of transition zone assembly" for publication in PLOS Biology. I have now obtained advice from the original reviewers and have discussed their comments with the Academic Editor. Based on the reviews (attached below), we will probably accept this manuscript for publication, assuming that you will modify the manuscript to address the remaining points raised by the reviewers. We would also like you to consider some suggestions to improve the title. 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We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: --------------------------------------------------------Reviewers’ commentsRev. 1:In this revised manuscript, Wu et al. largely address the concerns I raised previously. While it is unfortunate that the Cep290-Dzip1l interaction could not be dissected more precisely, the authors made a reasonable effort, and they revised the text accordingly. I would encourage the authors to include the coIP showing interaction of mammalian Dzip1l and Cep290 if possible. There are also some grammatical errors that should be corrected before publication, and parts of the Discussion are not as clearly written as the rest of the manuscript. These minor issues notwithstanding, I believe the manuscript is suitable for publication in PLoS Biology.Rev. 2:The authors have answered to all my initial requests and the additional data reinforce their conclusions.In particular they have corrected all the overstatements and added interesting data showing functional interactions between Fam92 and Rab8, indicating that Rab8 and Fam92 or Cby cooperate during spermatogenesis.Minor point:Page 13 fourth line: MKS6 is not presented on sup Fig 4C.page 14 last paragraph: "is still existed" check English grammar.Rev. 3:The revised manuscript has been improved and addressed most of my concerns. Though how the N-terminus of CEP290 is anchored to the basal body to recruit Dzip1 is not completely clear. Overall this work and model proposed for CEP290-Dzip1-Rab8 pathway in TZ assembly initiation is important to the field.Rev. 4:The revised work by Wu et al represents a significant genetic and biochemical study into early stages of ciliogenesis in Drosophila regulated by CEP290 through the DZIP1-CBY/Rab8 module. Using a range of imaging modalities and generating many genetic tools, the work is novel and sound- appropriate for publication in a revised manner in PLoS Biology.1. Accessibility: I am disappointed the authors did not choose to make their data more accessible to the broad readership of the journal. 10% of all readers, including some of the corresponding authors whose work they cite here, are red-green colorblind and as such are unable to interpret the bulk of the data presented in this manuscript. Changing pseudocolors in two color panels is very easy in FIJI with plug-ins- it would take the authors a half-day tops but would be so inclusive, improving pick up and citation of their work. .2. Allelism and expression: as I wrote, we know from human genetics that levels of variant expression are key for CEP290 phenotypes- this is ultimately at the protein level, which the authors are unable to look at. I think this statement needs to be made explicitly as a caveat to their interpretation in the discussion. Something akin to 'Without a robust antibody to survey expression to validate levels of CEP290 protein expression within our alleles, we cannot fully unpick how much of our phenotypes are due solely to reduced levels versus requirement for specific domains. We suspect it is likely a combination of both'. Reviewer 1 (Question 2) also raises issues of protein levels, as does reviewer 2 who wrote '…all the experiments based on the expression of these truncated forms, could there be an effect of the expression levels?' Their response 'To ensure that CEP290 and its truncations were expressed at a comparable level, all these transgenes were inserted o the same integration platform (the attP site of 25C6 locus on chromosome 2), and they were driven by the same ubiquitin promoter. Therefore, it is unlikely that the different defective phenotypes with expressing various CEP290 truncations are due to their different expression levels'. Again, this reflect only transcript level and not stability of the protein- this is emphasized in Supp Fig 8 where there is no GFP expressed for CEP290-M suggesting it is not stable. Without a GFP blot to show similar levels of protein expression, untangling protein instability/levels versus changes in localization cannot be unpicked.Minor points:1. A comment: My point about was about on-target 'off-targets' in their crispant alleles- larger or complex rearrangements at the locus that because they are at the locus and cannot be 'eliminated by outcrossing 3 times to dilute any potential off targeted lesion'. Large long read sequencing is necessary and/or southern blots to really confirm, but I suspect were not done.2. Last paragraph of the introduction- it is all past tense, but work you describe in the paper- present tense is more apropos.3. Figure 1C legend: Sequencing of what gDNA or cDNA? Also in this figure why are the numbers different at the bottom of the chromatograms- consider removing as they are confusing, without adding value.4. p7 'This is consistent with the transcription level of cep290 in cep290ΔC mutant' should read ' consistent with the transcript level'- you are assaying only transcript level (result of production and turnover).5. P8 Protein DILA should be all caps.6. Top of p12 typo : 'Drosophila DIZP1 is' should read 'Drosophila DZIP1 is'7. Page 12 Overexpression in a cell line does not constitute in vivo 'GFP-tagged CEP290 and HA-tagged DZIP1 in cultured Drosophila S2 cells'- removing 'also occurs in vivo,' without losing emphasis.8. Top of p16- typo 'cby or fam92 single mutants'9. Top of p18- typo single or plural- clarify 'Then we created a fam92 deletion mutants' 10 Dec 2020AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 16 Dec 2020Dear Dr. Wei,I am writing concerning your manuscript submitted to PLOS Biology, entitled “CEP290 is essential for the initiation of ciliary transition zone assembly.”We have now completed our final technical checks and have approved your submission for publication. You will shortly receive a letter of formal acceptance from the editor.Kind regards,PLOS Biology


Fabricated nanocomposite membranes were also studied for their efficacy in the removal of both monovalent (NaCl) and divalent salts (Na2SO4). The results revealed that the membrane possesses complete permeation to NaCl with less rejection of Na2SO4 (<5%). In addition, the nanocomposite membrane revealed outstanding antifouling performance with the flux recovery ratio (FRR) of 73% towards bovine serum albumin (BSA). Therefore, the in-house prepared novel nanocomposite membrane is a good candidate for the effective decolorization of wastewater containing dye.In this paper, novel zwitterionic graphene oxide (GO) nanohybrid was synthesized using monomers [2-(Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA) and N,N′-methylenebis(acrylamide) (MBAAm) (GO@poly(SBMA-co-MBAAm), and incorporated into polysulfone (PSF) hollow fiber membrane for the effectual rejection of dye from the wastewater. The synthesized nanohybrid was characterized using FT-IR, PXRD, TGA, EDX, TEM and zeta potential analysis. The occurrence of nanohybrid on the membrane matrix and the elemental composition were analyzed by XPS. The as-prepared tight ultrafiltration hollow fiber membrane exhibited high rejection of reactive black 5  and reactive orange 16  at a dye concentration of 10 ppm and pure water flux (PWF) of 49.6 L/m Among the organic dyes used, reactive dyes have gained significant attention owing to the low tendency of fixing on the fibers. Accordingly, a considerable amount of inorganic salts should be used to enhance the binding capability, which led to the existence of a large number of dyes and salts in the wastewater stream3. The discharge of untreated wastewater effluents into the environment, not only affect human beings but also restrict the permeation of light which will affect the aquatic flora and fauna to a greater extent8. Further, the discharged dye molecules are highly vulnerable to hydrolysis or oxidation to degrade into different toxic substances. Reactive dyes are capable of causing skin diseases like contact dermatitis and respiratory diseases like asthma9. In the matter of sustainability, dyes and inorganic salts should be separated from the wastewater and recycled rather than dye removal or water purification by reverse osmosis (RO). Additionally, the recycled dye can be used in the dyeing process and salts can work well as draw solution in forward osmosis (FO)12.In recent times, wastewater generated from the textile industries becoming detrimental environmentally demanding effluent. It is mainly composed of a large amount of organic dyes and inorganic salts, properly ~5.6 wt% of Na17. In the 1980s, the nanofiltration (NF) membrane was introduced and has become an attractive technology due to the improved selectivity, high permeation, low operating pressure and less maintenance cost21. NF membrane has a pore size of ~0.5–2.0 nm and molecular weight cut-off (MWCO) between 200 and 1000 Da, which renders the opportunity to stand superior in wastewater purification22. The mechanism of NF membrane separation is based on size exclusion and electrostatic repulsion (Donnan effect). However, NF membrane performances are severely affected by concentration polarization and membrane fouling23. Essentially, in textile wastewater treatment, the flux decline is triggered by the cake layer formation and pore blockage24. According to Koyuncu, membrane fouling can be reduced by increasing the cross-flow velocity25. In addition, the hydrophobic interaction and electrostatic attraction will also contribute to the reduced flux via the accumulation of dye molecules in the membrane pore structure26. Consequently, it increases the frequency of chemical cleaning, which reduces membrane life. Besides, the high concentration of inorganic salts reduces the permeate flux due to the increase in osmotic pressure27. Therefore, advanced NF membranes having dye removal capacity with low salt rejection and high flux are highly anticipated in the field of textile wastewater treatment.Membrane separation is one of the efficient and popular techniques for the purification of wastewater29. Lin et al. demonstrated the fractionation of dye/Na2SO4 using the UH004 T-UF membrane (MWCO 4700 Da) from textile wastewater. The results revealed thatmembrane could reject >98.9% of both reactive blue 2 and direct dyes along with the high permeation of Na2SO428. Liu et al. applied positively charged T-UF membrane with MWCO of 12700 Da for separation of dye, which allowed 99.9% rejection of Congo red with dye solution permeation of 84 L/m2h30. Hydrophilic polyaryletherketone (PAEK)-COOH T-UF membrane was reported by the Mao group. The prepared membrane exhibited rejection of 99.8% Congo red (100 ppm) with the complete permeation of NaCl and <10% rejection to Na2SO429. Hence, the T-UF membrane having both the high flux of water and inorganic salts with high rejection of dye molecules is highly desirable since the textile effluents increase day by day.The usage of tight ultrafiltration (T-UF) can be recognized as an operative approach for the bifurcation of dye/salt mixture34. Graphene oxide (GO) consists of reactive functional groups such as -OH, epoxy, and -COOH has designated as an effective substitute for constructing the nanocomposite membranes owing to its characteristic 2D structure, high chemical stability, strong hydrophilicity, and high surface area35. GO has been incorporated into the membrane matrix for constructing nanocomposite membranes with enhanced flux and antifouling properties38. Nevertheless, graphene derivatives are susceptible to form aggregation in dope solution, as a result, it forms a random dispersion. Furthermore, the compatibility between the membrane matrix and GO is poor, which may enhance the defects in the matrix. Consequently, there will be a compromise in the selectivity and mechanical strength of the nanocomposite membrane39. For that reason, surface modification of GO for the betterment of improved performance and compatibility is mandatory. Zwitterionic materials bearing both anionic and cationic groups have been developed as favorable antifouling and superhydrophilic materials. It establishes a strong hydration layer on the membrane surface, which is essential for reducing the adsorption of foulants capability and enhancement of flux40.The carbon-based nanomaterials have been demonstrated many interesting properties such as adsorption of organic dyes and heavy metals, desalination and antimicrobial activityet al. described the preparation of zwitterionic GO via reverse atom transfer radical polymerization (RATRP), which is overall in three steps1. Zhao et al. prepared zwitterionic GO via free radical polymerization at 60 °C under a nitrogen atmosphere. The prolonged reaction time (40 h) and inert atmosphere still increase the operational cost41. He et al. stated the functionalization of GO in two steps. In the first step, GO was vinylated by 3-(methacryloxy) propyltrimethoxysilane (MPS) followed by reaction with some of the monomers via surface-initiated precipitation polymerization42.The nanocomposite membrane will have improved compatibility, as the hydrophobic chains in the zwitterionic polymers are miscible with the polymer matrix. Zhu et al. for the preparation of poly(divinylbenzene) microspheres43. It is described that the size of particles can be altered by changing the concentration of the monomer and radical initiator. In addition, the increased degree of cross-linking also influences the size of the particles. DPP is i) Time-saving (<2 h) and facile method compared to the conventional polymerization process. ii) No stabilizer and pH adjustment are required. iii) As reaction proceeds at refluxing temperature, no need for the inert atmosphere. iv) Prepared particles show excellent colloidal stability in various solvents without adding any stabilizing agents. In the typical process, acetonitrile (ACN) was chosen as a reaction solvent, since the monomers and initiator are highly soluble and synthesized nanoparticles are insoluble. Xihao et al. described the preparation of functionalized Fe3O4 nanoparticles via DPP. The as-prepared hydrophilic nanoparticles demonstrated the improved binding capacity towards glycoproteins44. Guangwei et al. modified the MWCNTs with functional groups such as –COOH, –SO3H and PO3H2 by DPP. It was stated that the modified MWCNTs improved proton conductivity45.Distillation precipitation polymerization (DPP) is one of the developing methods for the preparation of uniform-sized micro and nanoparticles. It was first reported by Bai et al. reported the first paper on the surface functionalization of multiwalled carbon nanotube (MWCNTs) with poly(acrylic acid) and poly(acrylamide) via in situ radical polymerization46. Later, the GO surface was functionalized with poly(acrylic acid) and poly(acrylamide) via covalent bonding, which was reported by Mingxin et al.47. In 2011, Kan et al. demonstrated the functionalization of GO with different polymer chains via free radical polymerization48. It was stated that after functionalization, the aggregation of GO was reduced to a greater extent. In this typical reaction, polymer chains are directly attached to the C=C bond of GO and the rest of the active functional groups were intact. Further, by combining the advantages of this method and DPP would provide a simple and cost-effective methodology for the functionalization of GO. In this typical polymerization process, the vinyl monomers are initiated by radical polymerization and the formed radicals are added to C=C bonds of GO via propagation. Consequently, there is a formation of many radicals on the GO surface, which undergoes further chain propagation and termination. Conversely, so far no experimental evidence has been reported to support the above-said mechanism49. Additionally, the functional groups such as –COOH and –OH of GO are intact. Thus, there was no compromise in the hydrophilicity of GO after functionalization.Jianfeng Inspired by the above reports, GO@poly(SBMA-co-MBAAm) nanohybrid was synthesized via DPP using GO as the backbone, MBAAm as a cross-linking agent and SBMA as a zwitterionic monomer. This material was used as an additive to prepare tight ultrafiltration (T-UF) hollow fiber (HF) membranes, using polysulfone (PSF) as the membrane material. The presence of all the elements and membrane morphology were studied using XPS and SEM analyses. The effect of incorporated nanocomposite on T-UF HF membranes was examined based on the surface hydrophilicity, water uptake, MWCO, and pure water flux. The as-prepared membrane’s filtration performance was demonstrated for dye and salt separation using negatively charged reactive dyes as model pollutants in detail. Additionally, the dye rejection at different concentrations of dye and salt, different pH, various pressure and antifouling performances have also been studied. To the best of our understanding, this is the first such paper which describes the synthesis of novel GO@poly(SBMA-co-MBAAm) nanohybrid and used as an additive in PSF T-UF HF membrane.® P-1700) was purchased from Solvay chemicals. SBMA, RB-5, RO-16 and bovine serum albumin (BSA) were procured from Sigma Aldrich and the molecular structure of reactive dyes is depicted in Fig. 2SO4), sulfuric acid (H2SO4), sodium chloride (NaCl), hydrogen peroxide (H2O2), potassium permanganate (KMnO4), phosphoric acid (H3PO4) acquired from Merck.Azobisisobutyronitrile (AIBN), graphite powder, acetonitrile (ACN), N-methyl-2-pyrrolidone (NMP), polyvinylpyrrolidone (PVP) and MBAAm were obtained from Loba chemicals. Polysulfone pellets . The GO nanosheets were washed with water until the neutral pH and finally with 1 × 130 mL of ethanol and isolated by centrifugation.Graphene oxide (GO) was prepared as stated in the literatureThe GO@poly(SBMA-co-MBAAm) nanohybrid was synthesized via a one-step DPP technique. Typically, 0.1 g of GO was dispersed in 100 mL of acetonitrile in a dried round bottom flask by using an ultrasonic cleaning bath (Anmanm industries USC-100). Added 0.2 g (0.71 mmol) of SBMA, 0.8 g (5.18 mmol) of MBAAm and 0.02 g (0.12 mmol) of AIBN and stirred for 15 min at room temperature. The reaction mass was purged with nitrogen gas for 20 min to eliminate the dissolved oxygen. Later the reaction was performed by attaching Dean-Stark receiver and heated in an oil bath to 75 °C for another 15 min. The oil bath temperature was gradually rose to 110 °C to continue the polymerization reaction at reflux. When 40 mL of acetonitrile has removed over 40 min through Dean-Stark receiver, the reaction mixture allowed to room temperature and agitated for an additional 1 h. The nanoparticles were isolated and washed with acetonitrile to remove the oligomer and unreacted monomer. The material was dried at 55 °C for 15 h (Yield = 0.9 g). The synthetic route of GO@poly(SBMA-co-MBAAm) nanohybrid is represented in Scheme 1  concentration of 0, 0.1, 0.25 and 0.5 wt% from now denoted to as TM-0, TM-1, TM-2 and TM-3.The details of the characterization of nanohybrid and membranes are provided in the Supporting Information.PSF tight ultrafiltration (T-UF) hollow fiber (HF) membranes were prepared by the dry/wet phase inversion method. The PSF and PVP were dried at 60 °C for 24 h prior to dope solution preparation to eliminate the adsorbed water molecules. Details of the spinning parameter and dope solution composition are depicted in Tables −1 due to O-H stretching vibration and the C=O peak was observed at 1735 cm−1. The peaks at 1624, 1219 and 1056 cm−1 were owing to C=C, C-O, and C-O-C vibrations respectively52. ForGO@poly(SBMA-co-MBAAm), peaks at 3065 and 2947 were ascribed to C-H stretching vibrations. The ester group present in SBMA was observed at 1722cm−1 41. The amide group C=O stretching and N-H bending vibrations were detected at 1657 and 1530 cm−1. More importantly, peaks at 1043 and 1117 cm−1 were assigned to the symmetric and asymmetric stretching vibrations of the sulfonate (SO3−) group. The peak at 1209 cm−1 was bestowed by C-N stretching vibration53. In that way, the modification of GO was confirmed by FT-IR. Furthermore, TM-0 and TM-2 HF membrane ATR-FTIR spectra were also recorded and presented in Figure −1, 1242 cm−1 and 1150 cm−1 were attributed to the characteristic S=O asymmetric stretch, C-O-C stretch and S=O symmetric stretch of PSF. The peak at 1662 cm−1 in both TM-0 and TM-2 membrane due to amide C=O stretch of residual PVP. The new peak at 1734 cm−1 in TM-2 membrane was due to the C=O stretch (carboxylic acid group) of GO, which confirmed the presence of nanomaterial in the TM-2 matrix. In addition, there was no appreciable shift in stretching frequency was observed in addition to PSF characteristic peaks by adding GO@poly(SBMA-co-MBAAm), which indicates no interaction between GO@poly(SBMA-co-MBAAm) and PSF chain.GO and GO@poly(SBMA-co-MBAAm) were examined by FT-IR and are presented in Figure 54. In summary, the morphological changes in TEM images confirmed the functionalization of GO.The morphology of zwitterionic GO was observed in TEM and shown in Figure 56. However, in the case of GO@poly(SBMA-co-MBAAm), the [001] reflection of GO at 2θ = 9.8° was not detected as the regular stack of GO was demolished by the intercalation of nanosize poly(SBMA-co-MBAAm) and the increased disorder, which provides additional confirmation of intercalation of the poly(SBMA-co-MBAAm) at GO. The obtained result is well aligned with the literature61. Furthermore, the increased interlayer spacing and disorder of the GO@poly(SBMA-co-MBAAm) nanohybrid would have more amount of active sites than the GO.Figure 2 and steam release63 and slower weight loss between 450 and 900 °C was ascribed to the decomposition of more stable oxygen-containing functionalities64. In addition, the total weight loss of GO was ca. 65.1%. However, in the case of GO@poly(SBMA-co-MBAAm) the total weight loss was ca. 80.8%. The difference in weight loss validates the successful functionalization of GO. Therefore, the loading density of MBAAm and SBMA on GO was ca. 157 mg/g.TGA was used to understand the thermal properties of GO and GO@poly(SBMA-co-MBAAm) and presented in Figure 66. The prepared GO  are presented in Figure +/CSO3−, C-O/C-N, C=O, and O-C=O respectively. In Fig. 4N+with the binding energies of 400.48, 398.18 and 402.28 eV. The atomic % of all the elements present in the membrane are as follows. For TM-0 membrane, C - 83.10%, O - 12.21% and S - 4.69% and for TM-2 membrane, C - 81.85%, O - 13.88%, S - 2.84% and N - 1.44%. Therefore, XPS analysis confirms the occurrence of nanohybrid in the membrane matrix.Figure As tabulated in Table 70, which is necessary for reducing the maintenance cost and to increase the membrane lifecycle. Figure 71.The hydrophilicity of the membrane was measured by contact angle analysis. In general, the hydrophilic membrane surface is less prone to foulingTo understand the surface hydrophilicity further, water uptake capacity of the pristine and nanocomposite membrane was carried out and presented in Table 72. As explained above  and TM-2 membrane are presented in Figure e Figure , the [00The surface zeta potential of TM-0 and TM-2 T-UF HF membranes was analyzed as a function of pH and presented in Figure The MWCO of all the T-UF HF membranes was determined from the rejection of PEG with different molecular weights. As can be seen from Figure 2 release. The weight loss of around 500 °C in TM-0 and TM-2 HF membranes was attributed to the decomposition of the aromatic backbone in PSF.The effect of GO@poly(SBMA-co-MBAAm) nanomaterial on the thermal stability of the PSF HF membrane was analyzed by TGA. As shown in Figure 2h. However, in the case of the TM-2 membrane, PWF was improved to 49.6 L/m2h. This enhancement was attributed to the incorporation of nanohybrid, which provided improved hydrophilicity, porosity and slack structure of the nanocomposite membrane. The zwitterionic materials have a functional group such as sulfonate and a quaternary ammonium group, which has a strong attraction towards the water molecules via electrostatic attraction. Therefore, the attraction between the water molecules and the membrane surface will be more. Further, the incorporated nanohybrid in the polymer matrix can diminish the interaction of polymer chains to some extent. Therefore, it decreases the resistance to the transport of water molecules across the membrane and led to increased flux. Nevertheless, for the TM-3 membrane the PWF, hydrophilicity and porosity were decreased. The reason for this reduction is as follows. With a further increase of nanohybrid, tend to agglomerate and obstruct the pathway of permeation. As a result, PWF and porosity were reduced. The decreased hydrophilicity was due to the enhancement of hydrophobic alkyl groups in the MBAAm and SBMA. In summary, the as-prepared T-UF HF membrane noticeably exhibited an increased PWF compared to the pristine membrane.The pure water flux (PWF) of as-prepared membranes is represented in Table et al.30. The four charged RB-5 demonstrated the highest rejection than the two charged RO-16. In general, the higher the charge of the dye molecules, the greater would be the degree of hydration. Consequently, the size of the dye molecule increases. As shown in Figure −, SO42− and HCO3− in the feed solution. The presence of such ionic species would increase the electrostatic shielding effect, thereby decreasing the rejection of dyes12. A similar report had been published for the decreased rejection of direct red 2328. Meanwhile, the permeated dye molecules cause fouling, which results in decreased dye solution flux slightly.To evaluate the dye removal capacity of the as-prepared membrane, the rejection experiment was carried out with various concentrations of RB-5 and RO-16. Among all the prepared membranes, TM-2 was preferred for the dye removal studies. Figure et al.28 and Petrinic et al.73.The performance of the TM-2 membrane with respect to applied pressure is depicted in Figure 74, therefore, membrane flux decreases. Furthermore, the decreased rejection can be ascribed to the inferior electrostatic repulsion between the membrane surface and dyes. At high basic conditions, the electrostatic interaction is screened by the presence of OH–ions. It was noted that at pH 3, dye rejection was increased, and flux was declined. In acidic conditions, dye molecules get protonated to a greater extent, which in turn solubility of dye molecules are reduced and get precipitated in the feed tank. As a result, rejection increases owing to precipitation, while flux decreases because of membrane fouling from dye precipitation on the membrane surface. The feed and permeate digital photographic images of RB-5 and RO-16 is depicted in Figure The separation efficiency of membranes can be effectually altered by the solution pH since the electrochemical properties of the membrane surface and the dye molecules are directly affected. Figure 77. Subsequently, a membrane with high rejection of dye molecules with high salt permeation is highly needed to process the textile wastewater. TM-2 membrane exhibited complete permeability to NaCl and <5% rejection to Na2SO4. The filtration performance of the TM-2 membrane for RB-5 and RO-16 at different concentrations of Na2SO4 is illustrated in Figure et al. salt/dye solution flux was decreased with the increase of salt concentration. The reduced flux was attributed to the change in the osmotic pressure, which reduces the net driving pressure. In spite of the TM-2 membrane exhibits lower rejection for Na2SO4, water transports faster than salt (“Dilute effect”) due to steric exclusion78. However, the rejection was increased as the dye molecules form aggregates under the influence of added salt. Some of the recent literature reported and commercially available membranes are summarized in Table As textile wastewater is a mixture of dye and inorganic salts, it is important to study the rejection profile of salt and salt/dye mixture. Further, the added salt has a direct impact on membrane performancesFurthermore, a short-term stability study was performed and depicted in Figure 79, simple hydraulic cleanings will not help to recover the maximum membrane flux until the membrane has antifouling property. Hence, high flux, low fouling tendency, and high rejection capability are the prerequisites of the nanocomposite membrane. In the present study, BSA was used as a model protein. Generally, as the size of BSA molecules is larger than the membrane pore size, it adsorbs on the membrane surface rather than penetrate the pore. Thus, it causes severe fouling on the membrane surface.The membrane filtration performance greatly depends on the antifouling property. Typically, membranes used in wastewater treatment are more vulnerable to membrane fouling, which was ascribed to the adsorption and aggregation of foulants on the hydrophobic membrane surface. Consequently, membrane flux is reduced. As the foulants are contacting the membrane surface via hydrogen bonding, electrostatic interaction and weak van der Walls forceFigure t), reversible fouling (Rr) and irreversible fouling (Rir) were also calculated for better understanding and values are tabulated in Table 80. Thus, the loosely adsorbed foulants were easily removed with a simple hydraulic water wash. Moreover, since the as-prepared nanocomposite membrane is negatively charged at pH 7.4, it also would help to inhibit the adsorption of negatively charged BSA molecules through electrostatic repulsion. The Rt of the pristine membrane was 81.8%, which shows the inferior antifouling nature of TM-0. The nanocomposite membrane TM-2 demonstrated the Rir of 17.4%, which confirmed the loose adhesion of foulants on the membrane surface. In the case of TM-0, Rir was 38.8% as the foulants had a strong attraction towards the membrane surface. Still, TM-3 membrane performance was less compared to TM-2. The reason for the reduced performance of TM-3 could be attributed to the agglomeration of nanohybrid at the higher concentration. In summary, the antifouling property of the TM-2 membrane was significantly enriched.The calculation of the flux recovery ratio (FRR) would be useful to determine the antifouling performance of the membranes. In addition, total organic fouling (R2h) and reactive dyes such as RB-5 (99%) and RO-16 (74%) rejection with high salt flux, which was owing to the presence of nanohybrid. TM-2 membrane with 0.25 wt% loading of nanohybrid exhibited the Rir of 17.4%, signifying enhanced antifouling ability; the as-prepared novel tight UF hollow fiber membrane demonstrates a potential application for salt/dye mixture separation in textile wastewater.The prepared GO surface was functionalized with zwitterionic sulfobetaine via DPP. The as-synthesized nanohybrid was incorporated into the PSF membrane matrix and tight ultrafiltration hollow fiber membranes were prepared by dry/wet phase inversion method. The modification of GO was confirmed by spectroscopic and microscopic techniques. The existence of nanohybrid in the membrane matrix was investigated by XPS analysis. The impact of nanohybrid on the hydrophilicity, water uptake, porosity, MWCO, morphology, flux, and antifouling capacity of nanocomposite membranes were explored. The nanocomposite membrane exhibited high pure water flux (49.6 L/m